Detection and Quantitation of Benzo[a]pyrene-Derived DNA Adducts in Mouse Liver by Liquid Chromatography−Tandem Mass Spectrometry:  Comparison with ³²P-Postlabeling

Abstract

The polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) is a proven animal carcinogen that is potentially carcinogenic to humans. B[a]P is an ubiquitous environmental pollutant and is also present in tobacco smoke, coal tar, automobile exhaust emissions, and charred food. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using electrospray ionization and selected reaction monitoring (SRM) has been developed for the detection of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE-N(2)dG) adducts formed in DNA following the metabolic activation of B[a]P to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE). The method involves enzymatic digestion of the DNA sample to 2'-deoxynucleosides following the addition of a stable isotope internal standard, [(15)N(5)]B[a]PDE-N(2)dG, and then solid phase extraction to remove unmodified 2'-deoxynucleosides prior to analysis by LC-MS/MS SRM. The limit of detection of the method was 10 fmol (approximately 3 B[a]PDE-N(2)dG adducts per 10(8) 2'-deoxynucleosides) using 100 microg of calf thymus DNA as the matrix. Calf thymus DNA reacted with B[a]PDE in vitro and mouse liver DNA samples at different time points following dosing intraperitoneally with 50, 100, and 200 mg/kg B[a]P was analyzed. Three stereoisomers of the B[a]PDE-N(2)dG adduct were detected following the reaction of calf thymus DNA with B[a]PDE in vitro. The levels of B[a]PDE-N(2)dG DNA adducts in the mice livers were found to increase in a dose-dependent manner with adducts reaching maximal levels at 1-3 days and then gradually decreasing over time but still detectable after 28 days. A very good correlation (r = 0.962, p < 0.001) was observed between the results obtained for the mouse liver DNA samples using LC-MS/MS SRM as compared to those obtained using a (32)P-postlabeling method. However, the levels of adducts observed following (32)P-postlabeling using butanol enrichment were approximately 3.7-fold lower. The LC-MS/MS method allowed the more precise quantitation of DNA adduct levels that were structurally characterized, in addition to a reduction in the time taken to perform the analysis when compared with the (32)P-postlabeling method.

Ultra-performance liquid chromatography–tandem mass spectrometry for rapid and highly sensitive analysis of stereoisomers of benzo[a]pyrene diol epoxide–DNA adducts

“…m/z 570 → 454) can preclude the presence of BPDE tetrols and other interference substance, thus, only four stereoisomers of the adducted dG are needed to be resolved regardless of the other components. Because of the improved detection selectivity, it takes shorter time (20-50 min) for the stereoslective separation [21,22,30]. Due to large sample consumption (30-300 g DNA), the LC-MS based methods are often used for identification of new DNA damage and analysis of DNA adducts from cultured cells or animal tissues rather than human biomonitoring [19,31,32].…”

Detection and quantitation of N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine adducts in DNA using online column-switching liquid chromatography tandem mass spectrometry

“…The development of electrospray ionization (ESI) has permitted the coupling of liquid chromatography (LC) to mass spectrometry (MS), which has been used for the detection of numerous DNA adducts [26]. LC-mass spectrometry has made sufficient gains in sensitivity for the detection of DNA adducts resulting in it being a viable alternative to other detection methods such as 32 P-postlabelling and immunoassays, to provide more accurate and precise results through the use of stable isotope internal standards [27,28]. Recently, LC-tandem mass spectrometry (MS/MS) methods using deuterated stable isotope internal standards and off-line pre-purification of the PhIP-C8-dG adduct using solid phase extraction or online column-switching LC have been described for the detection and quantitation of adducts in cells and DNA treated in vitro with PhIP, and also in the liver and colon of rats treated in vivo with PhIP [20,22,[29][30][31].…”

Preparation, identification and analysis of stereoisomeric anti-benzo[a]pyrene diol epoxide–deoxyguanosine adducts using phenyl liquid chromatography with diode array, fluorescence and tandem mass spectrometry detection

“…A number of methods have been developed for characterization and/or detection of BPDE-DNA adducts, including capillary electrophoresis (CE)-laser-induced fluorescence (LIF) and CE-LIF immunoassays [10][11][12][13], accelerator mass spectrometry [14], liquid chromatography or capillary electrophoresis coupled to mass spectrometry [15][16][17][18][19][20][21], and 32 P-postlabelling [20,22] and spectroscopic analysis [23,24]. Among these methods, CE-LIF immunoassays display advantages of measuring trace levels of DNA adducts without digestion of DNA [10,11].…”

“…A large amount of pure single BPDE-adducted nucleosides, which are not commercially available, are also required to be prepared in individual laboratories to characterize the stereochemistry of BPDE-DNA adducts generated in vitro or in vivo. Previously, single adducted nucleosides were obtained by reaction of anti-BPDE with DNA, followed by enzymatic digestion, liquid-liquid or solid-phase extraction, and HPLC purification [19,20,23]. In these procedures, multiple enzymatic digestions involved are time-consuming (∼24-72 h), which may cause excess hydrolysis of the BPDE-nucleosides into BPDE tetrols.…”

“…Another disadvantage is the low yield of anti-BPDEdG in DNA except (+)-trans-anti-BPDE-dG [24]. To produce enough anti-BPDE-dG standards, a large amount of highly carcinogenic anti-BPDE is often required (1-10 mg) [19,20,23]. An alternative is to use the reaction of dGMP and anti-BPDE, which only requires one-step enzymatic digestion for preparation of anti-BPDE-dGs [25].…”